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1.
Ultrastruct Pathol ; 48(5): 422-437, 2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-39166773

RESUMO

Renal disease is a common cause of morbidity and mortality in patients with plasma cell dyscrasias. The serum-free light chain assay is used in patients, mostly older, with unexplained acute kidney injury to screen for potential myeloma cast nephropathy. This study consists of a systematic review of diagnostic features in myeloma cast nephropathy. The morphological features of tubular casts in patients with multiple myeloma have not been systematically analyzed. This study focuses on the morphology of these casts, emphasizing ultrastructural features, in a series of 23 patients with light chain ("myeloma") cast nephropathy and compared them with casts in 10 patients with various diseases. The immunofluorescence data were correlated with morphological findings to provide diagnostic assessments and practice guidelines. The ultrastructural features identified as diagnostic of casts associated with myeloma included: amyloid and crystals in the casts, multiple well-defined fracture planes forming a complex jigsaw puzzle arrangement of cast contents, indicative of the fragility of the immunoglobulin light chains involved, and reactive tubular cells lining the tubules with the casts. These features were seen in 95.2% of MCN cases and none of the casts in other renal conditions. Myeloma casts exhibited light chain monoclonality in a significant percentage of the MCN cases and often no staining for IgA or IgM. In contrast, the majority of non-myeloma casts stained for both kappa and lambda light chains, lgA, and lgM, and showed ultrastructurally a rather uniform finely to coarsely granular electron density occasionally admixed with cellular debris.


Assuntos
Cadeias Leves de Imunoglobulina , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/patologia , Mieloma Múltiplo/ultraestrutura , Idoso , Pessoa de Meia-Idade , Cadeias Leves de Imunoglobulina/análise , Masculino , Feminino , Imunofluorescência/métodos , Nefropatias/patologia , Idoso de 80 Anos ou mais , Microscopia Eletrônica/métodos , Adulto
2.
Bull Exp Biol Med ; 177(2): 281-286, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-39096449

RESUMO

Different methods for fixing biofilms of Staphylococcus aureus and Salmonella enterica for light and electron microscopy were compared. Paraformaldehyde fixation did not preserve biofilm integrity during dehydration; Ito-Karnovsky fixation revealed cell morphology, but did not preserve the matrix. Ruthenium red combined with aldehydes allowed the matrix to be preserved and visualized. An analysis of the ultrastructure of S. aureus and S. enterica cells in biofilms and suspensions at various fixations is presented. The ultrastructure of the biofilm matrix has been described.


Assuntos
Biofilmes , Salmonella enterica , Staphylococcus aureus , Biofilmes/crescimento & desenvolvimento , Staphylococcus aureus/fisiologia , Salmonella enterica/fisiologia , Salmonella enterica/ultraestrutura , Salmonella enterica/crescimento & desenvolvimento , Formaldeído , Fixadores/farmacologia , Fixadores/química , Microscopia Eletrônica , Polímeros
3.
Methods Mol Biol ; 2841: 189-197, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39115778

RESUMO

Macroautophagy, hereafter autophagy, plays a crucial role in the degradation of harmful or unwanted cellular components through a double-membrane autophagosome. Upon autophagosome fusion with the vacuole, the degraded materials are subsequently recycled to generate macromolecules, contributing to cellular homeostasis, metabolism, and stress tolerance in plants. A hallmark during autophagy is the formation of isolation membrane structure named as phagophore, which undergoes multiple steps to become as a complete double-membrane autophagosome. Methodologies have been developed in recent years to observe and quantify the autophagic process, which greatly advance knowledge of autophagosome biogenesis in plant cells. In this chapter, we will introduce two methods to dissect the autophagosome-related structures in the Arabidopsis plant cells, including the correlative light and electron microscopy, to map the ultrastructural feature of autophagosomal structures, and time-lapse imaging to monitor the temporal recruitment of autophagy machinery during autophagosome formation.


Assuntos
Arabidopsis , Autofagossomos , Autofagia , Células Vegetais , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Arabidopsis/metabolismo , Arabidopsis/ultraestrutura , Autofagia/fisiologia , Células Vegetais/metabolismo , Células Vegetais/ultraestrutura , Imagem com Lapso de Tempo/métodos , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Microscopia Eletrônica/métodos
4.
Methods Mol Biol ; 2841: 199-205, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39115779

RESUMO

Biomolecular condensates are triggered by multivalent interactions conferred by the intrinsically disordered regions and/or interacting domains within the constituents. While light microscopy has provided powerful tools to study the dynamics of intracellular condensates, electron microscopy (EM) gives more detailed insights into their ultrastructure and spatial connectivity with membrane system. In this chapter, we describe the methods for detecting the membraneless condensates in plant cells by high-pressure freezing -based EM coupled with immuno-gold labeling and correlative light electron microscopy techniques, which may benefit researchers in future studies.


Assuntos
Células Vegetais , Células Vegetais/ultraestrutura , Células Vegetais/metabolismo , Microscopia Eletrônica/métodos
5.
Nat Commun ; 15(1): 6648, 2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-39103318

RESUMO

Mapping neuronal networks is a central focus in neuroscience. While volume electron microscopy (vEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide molecular information to identify cell types or functions. We developed an approach that uses fluorescent single-chain variable fragments (scFvs) to perform multiplexed detergent-free immunolabeling and volumetric-correlated-light-and-electron-microscopy on the same sample. We generated eight fluorescent scFvs targeting brain markers. Six fluorescent probes were imaged in the cerebellum of a female mouse, using confocal microscopy with spectral unmixing, followed by vEM of the same sample. The results provide excellent ultrastructure superimposed with multiple fluorescence channels. Using this approach, we documented a poorly described cell type, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.


Assuntos
Córtex Cerebelar , Animais , Feminino , Camundongos , Córtex Cerebelar/metabolismo , Córtex Cerebelar/citologia , Córtex Cerebelar/ultraestrutura , Microscopia Confocal/métodos , Microscopia Eletrônica/métodos , Conectoma/métodos , Neurônios/metabolismo , Neurônios/ultraestrutura , Corantes Fluorescentes/química , Camundongos Endogâmicos C57BL , Citologia
6.
Curr Biol ; 34(17): 3881-3893.e5, 2024 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-39127048

RESUMO

Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and there has been progress in structural studies on recombinant subassemblies. However, there is limited structural information on native kinetochore architecture. To address this, we purified functional, native kinetochores from the thermophilic yeast Kluyveromyces marxianus and examined them by electron microscopy (EM), cryoelectron tomography (cryo-ET), and atomic force microscopy (AFM). The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with microtubules and locating the microtubule binder, Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies and provides the foundation to study the global architecture and functions of kinetochores at a structural level.


Assuntos
Microscopia Crioeletrônica , Cinetocoros , Kluyveromyces , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Kluyveromyces/metabolismo , Kluyveromyces/ultraestrutura , Microscopia de Força Atômica , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Microscopia Eletrônica
7.
Nature ; 632(8026): 911-920, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39143214

RESUMO

Allosteric modulation of protein function, wherein the binding of an effector to a protein triggers conformational changes at distant functional sites, plays a central part in the control of metabolism and cell signalling1-3. There has been considerable interest in designing allosteric systems, both to gain insight into the mechanisms underlying such 'action at a distance' modulation and to create synthetic proteins whose functions can be regulated by effectors4-7. However, emulating the subtle conformational changes distributed across many residues, characteristic of natural allosteric proteins, is a significant challenge8,9. Here, inspired by the classic Monod-Wyman-Changeux model of cooperativity10, we investigate the de novo design of allostery through rigid-body coupling of peptide-switchable hinge modules11 to protein interfaces12 that direct the formation of alternative oligomeric states. We find that this approach can be used to generate a wide variety of allosterically switchable systems, including cyclic rings that incorporate or eject subunits in response to peptide binding and dihedral cages that undergo effector-induced disassembly. Size-exclusion chromatography, mass photometry13 and electron microscopy reveal that these designed allosteric protein assemblies closely resemble the design models in both the presence and absence of peptide effectors and can have ligand-binding cooperativity comparable to classic natural systems such as haemoglobin14. Our results indicate that allostery can arise from global coupling of the energetics of protein substructures without optimized side-chain-side-chain allosteric communication pathways and provide a roadmap for generating allosterically triggerable delivery systems, protein nanomachines and cellular feedback control circuitry.


Assuntos
Regulação Alostérica , Peptídeos , Proteínas , Regulação Alostérica/efeitos dos fármacos , Cromatografia , Retroalimentação Fisiológica , Ligantes , Microscopia Eletrônica , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Multimerização Proteica/efeitos dos fármacos , Proteínas/química , Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Proteínas/ultraestrutura
8.
Methods Mol Biol ; 2831: 11-20, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39134840

RESUMO

Recent advancements in nano- and microfabrication techniques have led to the development of highly biomimetic patterned substrates able to guide neuronal sprouting, routing, elongation, and branching. Such substrates, recapitulating shapes and geometries found in the native brain, may pave the way toward the development of cell instructive paradigms able to guide morphogenesis at the neuron-material interface. In this scenario, high-resolution electron microscopy approaches, owing to their ability of discerning the details of neural morphogenesis at a nanoscale resolution, may play a crucial role in unravelling the fine ultrastructure of neurons interfacing with biomimetic structured substrates.


Assuntos
Materiais Biomiméticos , Neurônios , Neurônios/ultraestrutura , Neurônios/citologia , Neurônios/metabolismo , Materiais Biomiméticos/química , Animais , Biomimética/métodos , Microscopia Eletrônica/métodos
9.
Commun Biol ; 7(1): 796, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38951162

RESUMO

The highly complex structure of the brain requires an approach that can unravel its connectivity. Using volume electron microscopy and a dedicated software we can trace and measure all nerve fibers present within different samples of brain tissue. With this software tool, individual dendrites and axons are traced, obtaining a simplified "skeleton" of each fiber, which is linked to its corresponding synaptic contacts. The result is an intricate meshwork of axons and dendrites interconnected by a cloud of synaptic junctions. To test this methodology, we apply it to the stratum radiatum of the hippocampus and layers 1 and 3 of the somatosensory cortex of the mouse. We find that nerve fibers are densely packed in the neuropil, reaching up to 9 kilometers per cubic mm. We obtain the number of synapses, the number and lengths of dendrites and axons, the linear densities of synapses established by dendrites and axons, and their location on dendritic spines and shafts. The quantitative data obtained through this method enable us to identify subtle traits and differences in the synaptic organization of the samples, which might have been overlooked in a qualitative analysis.


Assuntos
Microscopia Eletrônica , Fibras Nervosas , Sinapses , Animais , Camundongos , Microscopia Eletrônica/métodos , Fibras Nervosas/ultraestrutura , Sinapses/ultraestrutura , Axônios/ultraestrutura , Dendritos/ultraestrutura , Encéfalo/ultraestrutura , Córtex Somatossensorial/ultraestrutura , Camundongos Endogâmicos C57BL , Masculino , Software , Hipocampo/ultraestrutura , Hipocampo/citologia , Microscopia Eletrônica de Volume
10.
Methods Mol Biol ; 2827: 71-84, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38985263

RESUMO

The success of in vitro cultivation, particularly for micropropagation purposes, depends on the efficient control of contaminants. In this context, the sterilization of plant material constitutes a fundamental step in initiating cultures. Microbial contaminants can be found either on the surface (epiphyte) or inside plant explants (endophyte). However, the latter is generally challenging to detect and may not always be eradicated through surface sterilization alone. Endophyte contaminants, such as bacteria, can persist within plant material over several cultivation cycles, potentially interfering with or inhibiting in vitro establishment, growth, or recovery of cryopreserved materials. Therefore, microscopy techniques, such as electron microscopy, can yield valuable insights into bacterial endophytes' localization, tissue colonization patterns, and functions in in vitro plant culture. This information is essential for adopting effective strategies for eliminating, preventing, or harmonious coexistence with contaminants.


Assuntos
Bactérias , Endófitos , Microscopia Eletrônica/métodos , Plantas/microbiologia
11.
J Gen Physiol ; 156(9)2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-38980209

RESUMO

Skeletal muscle, the major processor of dietary glucose, stores it in myriad glycogen granules. Their numbers vary with cellular location and physiological and pathophysiological states. AI models were developed to derive granular glycogen content from electron-microscopic images of human muscle. Two UNet-type semantic segmentation models were built: "Locations" classified pixels as belonging to different regions in the cell; "Granules" identified pixels within granules. From their joint output, a pixel fraction pf was calculated for images from patients positive (MHS) or negative (MHN) to a test for malignant hyperthermia susceptibility. pf was used to derive vf, the volume fraction occupied by granules. The relationship vf (pf) was derived from a simulation of volumes ("baskets") containing virtual granules at realistic concentrations. The simulated granules had diameters matching the real ones, which were measured by adapting a utility devised for calcium sparks. Applying this relationship to the pf measured in images, vf was calculated for every region and patient, and from them a glycogen concentration. The intermyofibrillar spaces and the sarcomeric I band had the highest granular content. The measured glycogen concentration was low enough to allow for a substantial presence of non-granular glycogen. The MHS samples had an approximately threefold lower concentration (significant in a hierarchical test), consistent with earlier evidence of diminished glucose processing in MHS. The AI models and the approach to infer three-dimensional magnitudes from two-dimensional images should be adaptable to other tasks on a variety of images from patients and animal models and different disease conditions.


Assuntos
Glicogênio , Músculo Esquelético , Humanos , Glicogênio/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/diagnóstico por imagem , Inteligência Artificial , Microscopia Eletrônica/métodos
12.
Adv Kidney Dis Health ; 31(3): 216-222, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-39004461

RESUMO

Membranoproliferative glomerulonephritis (MPGN) is no longer a disease but a pattern of injury in various diseases. Characterized by electron-dense deposits, mesangial proliferation, and duplication of the glomerular basement membrane, MPGN was previously classified by findings seen by electron microscopy. However, recognizing complement dysfunction in relation to cases with the MPGN pattern of injury substantially changed our view of its pathogenesis. A new classification, including immune complex-mediated and complement-mediated MPGN, has become preferable and has been adopted by international guidelines. Despite these advancements, accurate diagnosis of MPGN remains a clinical challenge, given the pathological and clinical similarities between immune complex-mediated and complement-mediated MPGN. Additional testing, such as molecular and genetic testing, is often necessary. Here, we will summarize our current understanding of the MPGN pattern of injury from a pathology perspective as an introductory article in the following chapters.


Assuntos
Glomerulonefrite Membranoproliferativa , Humanos , Glomerulonefrite Membranoproliferativa/patologia , Glomerulonefrite Membranoproliferativa/imunologia , Glomerulonefrite Membranoproliferativa/diagnóstico , Microscopia Eletrônica , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/imunologia , Membrana Basal Glomerular/patologia , Membrana Basal Glomerular/imunologia , Glomérulos Renais/patologia , Glomérulos Renais/imunologia
14.
Nature ; 631(8020): 360-368, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38926570

RESUMO

A deep understanding of how the brain controls behaviour requires mapping neural circuits down to the muscles that they control. Here, we apply automated tools to segment neurons and identify synapses in an electron microscopy dataset of an adult female Drosophila melanogaster ventral nerve cord (VNC)1, which functions like the vertebrate spinal cord to sense and control the body. We find that the fly VNC contains roughly 45 million synapses and 14,600 neuronal cell bodies. To interpret the output of the connectome, we mapped the muscle targets of leg and wing motor neurons using genetic driver lines2 and X-ray holographic nanotomography3. With this motor neuron atlas, we identified neural circuits that coordinate leg and wing movements during take-off. We provide the reconstruction of VNC circuits, the motor neuron atlas and tools for programmatic and interactive access as resources to support experimental and theoretical studies of how the nervous system controls behaviour.


Assuntos
Conectoma , Drosophila melanogaster , Neurônios Motores , Tecido Nervoso , Vias Neurais , Sinapses , Animais , Feminino , Conjuntos de Dados como Assunto , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/citologia , Drosophila melanogaster/fisiologia , Drosophila melanogaster/ultraestrutura , Extremidades/fisiologia , Extremidades/inervação , Holografia , Microscopia Eletrônica , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Neurônios Motores/ultraestrutura , Movimento , Músculos/inervação , Músculos/fisiologia , Tecido Nervoso/anatomia & histologia , Tecido Nervoso/citologia , Tecido Nervoso/fisiologia , Tecido Nervoso/ultraestrutura , Vias Neurais/citologia , Vias Neurais/fisiologia , Vias Neurais/ultraestrutura , Sinapses/fisiologia , Sinapses/ultraestrutura , Tomografia por Raios X , Asas de Animais/inervação , Asas de Animais/fisiologia
15.
J Assoc Res Otolaryngol ; 25(4): 341-354, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38937328

RESUMO

To enable nervous system function, neurons are powered in a use-dependent manner by mitochondria undergoing morphological-functional adaptation. In a well-studied model system-the mammalian cochlea, auditory nerve fibers (ANFs) display distinct electrophysiological properties, which is essential for collectively sampling acoustic information of a large dynamic range. How exactly the associated mitochondrial networks are deployed in functionally differentiated ANFs remains scarcely interrogated. Here, we leverage volume electron microscopy and machine-learning-assisted image analysis to phenotype mitochondrial morphology and distribution along ANFs of full-length in the mouse cochlea inner spiral bundle. This reveals greater variance in mitochondrial size with increased ANF habenula to terminal path length. Particularly, we analyzed the ANF terminal-residing mitochondria, which are critical for local calcium uptake during sustained afferent activities. Our results suggest that terminal-specific enrichment of mitochondria, in addition to terminal size and overall mitochondrial abundance of the ANF, correlates with heterogenous mitochondrial contents of the terminal.


Assuntos
Nervo Coclear , Mitocôndrias , Animais , Mitocôndrias/ultraestrutura , Camundongos , Nervo Coclear/ultraestrutura , Nervo Coclear/fisiologia , Microscopia Eletrônica , Fibras Nervosas/ultraestrutura , Masculino , Camundongos Endogâmicos C57BL
16.
Cardiovasc Pathol ; 73: 107670, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38880163

RESUMO

Electron microscopy (EM) was a popular diagnostic tool in the 1970s and early 80s. With the adoption of newer, less expensive techniques, such as immunohistochemistry, the role of EM in diagnostic surgical pathology has dwindled substantially. Nowadays, even in academic centers, EM interpretation is relegated to renal pathologists and the handful of (aging) pathologists with experience using the technique. As such, EM interpretation is truly arcane-understood by few and mysterious to many. Nevertheless, there remain situations in which EM is the best or only ancillary test to ascertain a specific diagnosis. Thus, there remains a critical need for the younger generation of surgical pathologists to learn EM interpretation. Recognizing this need, cardiac EM was made the theme of the Cardiovascular Evening Specialty Conference at the 2023 United States and Canadian Academy of Pathology (USCAP) annual meeting in New Orleans, Louisiana. Each of the speakers contributed their part to this article, the purpose of which is to review EM as it pertains to myocardial tissue and provide illustrative examples of the spectrum of ultrastructural cardiac pathology seen in storage/metabolic diseases, cardiomyopathies, infiltrative disorders, and cardiotoxicities.


Assuntos
Cardiopatias , Microscopia Eletrônica , Miocárdio , Humanos , Miocárdio/patologia , Miocárdio/ultraestrutura , Cardiopatias/patologia , Valor Preditivo dos Testes , Prognóstico , Animais
17.
Acta Neuropathol Commun ; 12(1): 88, 2024 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-38840253

RESUMO

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the coding sequence of huntingtin protein. Initially, it predominantly affects medium-sized spiny neurons (MSSNs) of the corpus striatum. No effective treatment is still available, thus urging the identification of potential therapeutic targets. While evidence of mitochondrial structural alterations in HD exists, previous studies mainly employed 2D approaches and were performed outside the strictly native brain context. In this study, we adopted a novel multiscale approach to conduct a comprehensive 3D in situ structural analysis of mitochondrial disturbances in a mouse model of HD. We investigated MSSNs within brain tissue under optimal structural conditions utilizing state-of-the-art 3D imaging technologies, specifically FIB/SEM for the complete imaging of neuronal somas and Electron Tomography for detailed morphological examination, and image processing-based quantitative analysis. Our findings suggest a disruption of the mitochondrial network towards fragmentation in HD. The network of interlaced, slim and long mitochondria observed in healthy conditions transforms into isolated, swollen and short entities, with internal cristae disorganization, cavities and abnormally large matrix granules.


Assuntos
Modelos Animais de Doenças , Doença de Huntington , Imageamento Tridimensional , Mitocôndrias , Animais , Doença de Huntington/patologia , Doença de Huntington/genética , Doença de Huntington/metabolismo , Mitocôndrias/ultraestrutura , Mitocôndrias/patologia , Mitocôndrias/metabolismo , Imageamento Tridimensional/métodos , Camundongos , Camundongos Transgênicos , Encéfalo/patologia , Encéfalo/ultraestrutura , Encéfalo/metabolismo , Microscopia Eletrônica/métodos , Masculino , Neurônios/patologia , Neurônios/ultraestrutura , Neurônios/metabolismo
18.
ACS Sens ; 9(6): 3048-3056, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38830243

RESUMO

Ribosomal RNA (rRNA) plays a vital role in binding amino acids together, which dictates the primary structure of a protein. Visualization of its intracellular distribution and dynamics during protein synthesis enables a better understanding of the correlated biological essence. However, appropriate tools targeting live cell rRNA that are capable of multimodal imaging at the nanoscale are still lacking. Here, we rationally designed a series of terpyridine ammonium iridium(III) complexes, one of which is capable of selectively labeling rRNA in living cells. Its metal core and photostable nature allow further super-resolution STED imaging of rRNA found on the rough endoplasmic reticulum at a ∼40 nm resolution that is well correlated under correlative light and electron microscopy (CLEM). Interestingly, the Ir(III) complex demonstrated rRNA dynamics in living cells while boosting protein synthesis at the nanoscale. Our work offers a versatile tool to visualize rRNA synchronously under optical and electron microscopy, which provides a better understanding of rRNA evolution in living systems.


Assuntos
Irídio , Piridinas , RNA Ribossômico , Irídio/química , RNA Ribossômico/química , Humanos , Piridinas/química , Complexos de Coordenação/química , Microscopia Eletrônica/métodos , Células HeLa , Imagem Óptica/métodos
19.
J Vis Exp ; (207)2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38856226

RESUMO

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds from time to time. In hemostasis, self-limiting platelet aggregation leads to the formation of a structured thrombus in which bleeding cessation comes from capping the hole from the outside. Detailed characterization of this structure could lead to distinctions between hemostasis and thrombosis, a case of excessive platelet aggregation leading to occlusive clotting. An imaging-based approach to puncture wound thrombus structure is presented here that draws upon the ability of thin-section electron microscopy to visualize the interior of hemostatic thrombi. The most basic step in any imaging-based experimental protocol is good sample preparation. The protocol provides detailed procedures for preparing puncture wounds and platelet-rich thrombi in mice for subsequent electron microscopy. A detailed procedure is given for in situ fixation of the forming puncture wound thrombus and its subsequent processing for staining and embedding for electron microscopy. Electron microscopy is presented as the end imaging technique because of its ability, when combined with sequential sectioning, to visualize the details of the thrombus interior at high resolution. As an imaging method, electron microscopy gives unbiased sampling and an experimental output that scales from nanometer to millimeters in 2 or 3 dimensions. Appropriate freeware electron microscopy software is cited that will support wide-area electron microscopy in which hundreds of frames can be blended to give nanometer-scale imaging of entire puncture wound thrombi cross-sections. Hence, any subregion of the image file can be placed easily into the context of the full cross-section.


Assuntos
Microscopia Eletrônica , Trombose , Animais , Camundongos , Microscopia Eletrônica/métodos , Trombose/patologia , Hemostasia , Punções/métodos
20.
J Vis Exp ; (207)2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38856213

RESUMO

Volume electron microscopy (Volume EM) has emerged as a powerful tool for visualizing the 3D structure of cells and tissues with nanometer-level precision. Within the retina, various types of neurons establish synaptic connections in the inner and outer plexiform layers. While conventional EM techniques have yielded valuable insights into retinal subcellular organelles, their limitation lies in providing 2D image data, which can hinder accurate measurements. For instance, quantifying the size of three distinct synaptic vesicle pools, crucial for synaptic transmission, is challenging in 2D. Volume EM offers a solution by providing large-scale, high-resolution 3D data. It is worth noting that sample preparation is a critical step in Volume EM, significantly impacting image clarity and contrast. In this context, we outline a sample preparation protocol for the 3D reconstruction of photoreceptor axon terminals in the retina. This protocol includes three key steps: retina dissection and fixation, sample embedding processes, and selection of the area of interest.


Assuntos
Retina , Retina/ultraestrutura , Animais , Microscopia Eletrônica/métodos , Imageamento Tridimensional/métodos , Camundongos , Microscopia Eletrônica de Volume
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